冷大气压等离子体(CAP)已成为治疗细菌感染的常规抗生素的潜在替代品或佐剂。包括由抗生素抗性病原体引起的。在这项研究中,研究了亚致死CAP暴露与常规抗菌剂协同根除铜绿假单胞菌生物膜的潜力。评估了在铜绿假单胞菌生物膜中亚致死CAP预处理之后或不存在的抗微生物剂的功效。CAP预处理导致所有三个测试菌株(PAO1,PA14和PA10548)的浮游和生物膜抗菌敏感性增加。具有单个抗菌剂的最小抑制浓度(MIC)和最小生物膜根除浓度(MBEC),在CAP预处理生物膜后显着降低(环丙沙星/庆大霉素降低512倍;妥布霉素降低256倍)。在使用的所有浓度的抗菌剂下,亚致死CAP暴露和抗微生物药物的组合在增加高峰代谢时间方面是有效的,通过等温微量热法测量,再次表明敏感性增强。已知CAP通过原位产生活性氧和氮物质(RONS)引起氧化应激而损伤细菌细胞膜和DNA。虽然确切的机制尚不清楚,外膜蛋白的氧化应激被认为会损伤/扰乱细胞膜,通过ATP和LDH泄漏证实,允许抗菌剂更有效地穿透细菌细胞,从而增加细菌的敏感性。转录组分析,揭示冷等离子体介导的氧化应激导致铜绿假单胞菌超氧化物歧化酶上调,cbb3氧化酶,过氧化氢酶,和过氧化物酶,以及反硝化基因的上调,表明铜绿假单胞菌使用这些酶降解RONS并减轻冷血浆介导的氧化应激的影响。CAP治疗还导致铜绿假单胞菌中信号分子ppGpp的产生增加,表明正在建立严格的回应。虽然我们没有直接测量持久细胞的形成,这种严格的反应可能与生物膜培养物中持久细胞的形成有关.ppGpp和多磷酸盐的产生可能与蛋白质合成抑制和增加外排泵活性有关,可能导致抗菌药物耐受性的因素。转录组学分析还显示,治疗后6小时,核糖体调节因子有下调,参与持久细胞的形成,表明细胞已经开始复苏/恢复。此外,暴露后4小时的CAP治疗引起毒力因子pyoverdine和pygiyanin的下调;暴露后6小时,毒力因子产量正在增加。转录组学分析提供了对铜绿假单胞菌生物膜表现出对抗菌剂的敏感性增强的机制的有价值的见解。总的来说,这些发现表明,第一次,短CAP亚致死预处理可以是增强铜绿假单胞菌生物膜对抗菌药物敏感性的有效策略,并为冷等离子体抗菌协同作用提供了重要的机制见解。转录组学分析的反应,和恢复,铜绿假单胞菌生物膜中的亚致死冷等离子体暴露改善了我们目前对冷等离子体生物膜相互作用的理解。
Cold atmospheric-pressure plasma (CAP) has emerged as a potential alternative or adjuvant to conventional antibiotics for the treatment of bacterial infections, including those caused by antibiotic-resistant pathogens. The potential of sub-lethal CAP exposures to synergise conventional antimicrobials for the eradication of Pseudomonas aeruginosa biofilms is investigated in this study. The efficacy of antimicrobials following or in the absence of sub-lethal CAP pre-treatment in P. aeruginosa biofilms was assessed. CAP pre-treatment resulted in an increase in both planktonic and biofilm antimicrobial sensitivity for all three strains tested (PAO1, PA14, and PA10548), with both minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) of individual antimicrobials, being significantly reduced following CAP pre-treatment of the biofilm (512-fold reduction with ciprofloxacin/gentamicin; and a 256-fold reduction with tobramycin). At all concentrations of antimicrobial used, the combination of sub-lethal CAP exposure and antimicrobials was effective at increasing time-to-peak metabolism, as measured by isothermal microcalorimetry, again indicating enhanced susceptibility. CAP is known to damage bacterial cell membranes and DNA by causing oxidative stress through the in situ generation of reactive oxygen and nitrogen species (RONS). While the exact mechanism is not clear, oxidative stress on outer membrane proteins is thought to damage/perturb cell membranes, confirmed by ATP and LDH leakage, allowing antimicrobials to penetrate the bacterial cell more effectively, thus increasing bacterial susceptibility. Transcriptomic analysis, reveals that cold-plasma mediated oxidative stress caused upregulation of P. aeruginosa superoxide dismutase, cbb3 oxidases, catalases, and peroxidases, and upregulation in denitrification genes, suggesting that P. aeruginosa uses these enzymes to degrade RONS and mitigate the effects of cold plasma mediated oxidative stress. CAP treatment also led to an increased production of the signalling molecule ppGpp in P. aeruginosa, indicative of a stringent response being established. Although we did not directly measure persister cell formation, this stringent response may potentially be associated with the formation of persister cells in biofilm cultures. The production of ppGpp and polyphosphate may be associated with protein synthesis inhibition and increase efflux pump activity, factors which can result in antimicrobial tolerance. The transcriptomic analysis also showed that by 6 h post-treatment, there was downregulation in ribosome modulation factor, which is involved in the formation of persister cells, suggesting that the cells had begun to resuscitate/recover. In addition, CAP treatment at 4 h post-exposure caused downregulation of the virulence factors pyoverdine and pyocyanin; by 6 h post-exposure, virulence factor production was increasing. Transcriptomic analysis provides valuable insights into the mechanisms by which P. aeruginosa biofilms exhibits enhanced susceptibility to antimicrobials. Overall, these findings suggest, for the first time, that short CAP sub-lethal pre-treatment can be an effective strategy for enhancing the susceptibility of P. aeruginosa biofilms to antimicrobials and provides important mechanistic insights into cold plasma-antimicrobial synergy. Transcriptomic analysis of the response to, and recovery from, sub-lethal cold plasma exposures in P. aeruginosa biofilms improves our current understanding of cold plasma biofilm interactions.